Thermosetting Neutralized Chitosan Composition Forming a Hydrogel, Lyophilizate, and Processes for Producing the Same

ABSTRACT

The aqueous thermosetting neutralized chitosan composition, forming a phosphate-free transparent hydrogel at a temperature higher than 5° C., comprises 0.1 to 5.0 w/w %, based on the total composition, of a reacetylated chitosan having a molecular weight of not smaller than 100 kDa and a deacetylation degree of 40 to 70%, neutralized with an hydroxylated base, and 1 to 30 w/w %, based on the total composition, of a complexing agent selected from polyoses and polyols derived from polyoses. Said composition is useful for the preparation of an injectable formulation.

FIELD OF THE INVENTION

The present invention relates to an aqueous thermosetting neutralizedchitosan composition forming a phosphate-free, transparent hydrogel at atemperature higher than 5° C., and to a process for producing the same.

Further, the present invention relates to a lyophilizate obtained byfreeze-drying the thermosetting neutralized chitosan composition of thepresent invention and to a process for producing the same.

BACKGROUND OF THE INVENTION

Hydrogels are attractive for biomedical applications.

Further, hydrogels exhibiting the specific ability of increasing theirviscosity with temperature, also called“thermosensitive/thermoresponsive/pseudothermosetting/thermogellinghydrogels”, were proved to have a facilitated application combined withan increased residence time at the site of application, and thereforemay be advantageously used for drug delivery or tissue augmentation.

As known from O. Felt et al. in The Encyclopedia of Controlled DrugDelivery, 1999, said thermosensitive hydrogels may be basedadvantageously on polymers of natural origin, for example on chitosanwhich is a commercially available inexpensive polymer derived fromchitin, the second most abundant polysaccharide after cellulose.

Chitosan is known as a chitin derivative obtained by partial tosubstantial alkaline N-deacetylation of chitin also namedpoly(N-acetyl-D-glucosamine), which is a naturally occurring biopolymer.

Chitosan contains free amine (—NH₂) groups and may be characterized asto the proportion of N-acetyl-D-glucosamine units and D-glucosamineunits, and such is expressed as the degree of deacetylation (DD) of thefully acetylated polymer chitin.

Parameters of chitosan influencing important properties such assolubility and viscosity are the degree of deacetylation (DD) which maybe understood as representing the percentage of deacetylated monomers,and the molecular weight (Mw).

Chitosan is known to be biodegradable, biocompatible, bioadhesive,bacteriostatic, and further to promote wound-healing, drug absorption,and tissue reconstruction.

Due to its above mentioned intrinsic properties, chitosan is known tohave numerous cosmetic and pharmaceutical activities, and has been alsowidely explored for various applications through gels.

Therefore, considering the advantageous properties of chitosan, there isa continuous need to improve the properties of known thermosensitivechitosan hydrogels which are still considered as very promising for awider range of biomedical applications.

WO-A-99/07416 (BIOSYNTHEC) discloses a pH-dependenttemperature-controlled chitosan hydrogel which has thermosensitiveproperties at neutral pH such that it has low viscosity at lowtemperature but gels at body temperature.

This thermosensitive chitosan hydrogel is prepared by neutralizing acommercial chitosan having a deacetylation degree of about 80% withmono-phosphate dibasic salts of polyols or sugars exemplified inparticular by β-glycerophosphate (β-GP).

However, presence of β-GP in the hydrogel leads to the followingdisadvantages.

β-GP is a negatively charged entity that can react with a positivelycharged bioactive component, leading to its precipitation or to thedisturbance of its liberation from the hydrogel.

Therefore, presence of β-GP renders chitosan/β-GP hydrogelsinappropriate for use with numerous drugs.

Further, the properties of this hydrogel, such as gelation time andviscosity, depend on the concentration of β-GP and are therefore limitedby the solubility of β-GP.

In particular, a high concentration of β-GP is required to have a lowgelation time avoiding the rapid elimination of the hydrogel after itsadministration.

However, a high concentration of β-GP also decreases the mechanicalproperties of the hydrogel.

Therefore, the gelation time has to be balanced with the consistency ofthe hydrogel, and it is not possible to obtain gels that have both a lowgelation time and a high viscosity, which would be a desirablecombination of characteristics.

Also, a too high concentration of β-GP may induce the precipitation ofthe hydrogel at its administration site.

Further, said thermosensitive chitosan/β-GP hydrogels were found to beturbid, thus rendering their use inappropriate for particularapplications such as ocular or topical administrations.

In addition, phosphate-containing materials may be inappropriate interms of biocompatibility (G. Molinaro et al., Biomaterials,23:2717-2722 (2002)).

In order to overcome the disadvantages of chitosan/β-GP hydrogels, itwas proposed in WO-A-2005/097871 (UNIVERSITE DE GENEVE) a thermosettingneutralized chitosan composition forming a phosphate-free transparenthydrogel at a temperature higher than 5° C., said composition comprisingan homogeneously reacetylated chitosan having a molecular weight of notsmaller than 200 kDa, and a deacetylation degree of 30-60%, neutralizedwith an hydroxylated base.

WO-A-2005/097871 also disclose that said composition may furthercomprise 1,3-propanediol to modulate the viscoelastic properties of thehydrogel.

However, 1,3-propanediol is neither mentioned as “generally recognizedas safe” (GRAS), nor recognized as an additive mentioned in USpharmacopoeia, European pharmacopoeia or Japanese pharmacopoeia so thatits use is limited in biomedical applications.

Further, thermosetting neutralized chitosan compositions containing1,3-propanediol loose their thermoresponsive properties uponlyophilization.

In view of the continuous heed to provide improved thermosensitivechitosan hydrogels for biomedical applications, the present inventorshave continued their researches to overcome the disadvantages of theknown thermosensitive hydrogels.

An object of the present invention is to provide an aqueousthermosetting neutralized chitosan composition forming a phosphate-freetransparent hydrogel having improved properties and being acceptable forbiomedical applications.

Another object of the present invention is to provide an aqueousthermosetting neutralized chitosan composition which may be storedeasily and which preserves its thermogelling properties after storage.

Still another object of the present invention is to provide an aqueousthermosetting neutralized chitosan composition having a facilitatedapplication, for example by injection using needles or minimallyinvasive techniques.

These objects are achieved by the present invention.

SUMMARY OF THE INVENTION

According to a first aspect, the present invention provides an aqueousthermosetting neutralized chitosan composition (herein, also called“composition of the present invention”) as defined in independent claim1 and its dependent claims 2-11.

According to a second aspect, the present invention provides alyophilizate of the aqueous thermosetting composition according to thefirst aspect, as defined in independent claim 12.

According to a third aspect, the present invention provides a processfor producing the aqueous thermosetting neutralized chitosan compositionaccording to the first aspect, as defined in independent claim 13 andits dependent claims 14-15.

According to a fourth aspect, the present invention provides a processfor producing the lyophilizate according to the second aspect, asdefined in independent claim 16.

According to a fifth aspect, the present invention provides the use ofthe composition according to the first aspect, or lyophilizate accordingto the second aspect, as defined in independent claims 17-20.

According to the present invention, adding a complexing agent selectedfrom polyoses and polyols derived from polyoses to a specified chitosancomposition allows advantageously to provide a phosphate-freetransparent hydrogel having improved properties, which is acceptable forbiomedical applications and which can be easily stored.

Other advantages of the present invention will appear in the followingdescription.

The present invention will be now described in a more detailed manner.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a device for measuring the injectability of the compositionof the present invention.

FIG. 2 shows the evolution of the elastic modulus G′ (storage modulus)and of the viscous modulus G″ (loss modulus) of the transparent hydrogelcontaining trehalose obtained in Example 1, as compared with the samehydrogel without trehalose, as a function of time when the temperatureincreases from 4 to 37° C.

FIG. 3 shows the evolution of the elastic modulus G′ (storage modulus)and of the viscous modulus G″ (loss modulus) of the transparent hydrogelcontaining trehalose obtained in Example 1, after preparation (A) andafter thawing (B), as a function of time when the temperature increasesfrom 4 to 37° C.

FIG. 4 shows the evolution of the elastic modulus G′ (storage modulus)and of the viscous modulus G″ (loss modulus) of the transparent hydrogelcontaining trehalose obtained in Example 1, after freeze-drying andreconstitution, as compared with the same hydrogel without trehalose, asa function of time when the temperature increases from 4 to 37° C.

FIG. 5 shows the evolution of the elastic modulus G′ (storage modulus)and of the viscous modulus G″ (loss modulus) of the transparent hydrogelcontaining trehalose obtained in Example 4, as compared with the samehydrogel without trehalose, as a function of time when temperatureincreases from 4 to 37° C.

FIG. 6 shows the evolution of the elastic modulus G′ (storage modulus)and of the viscous modulus G″ (loss modulus) of the transparent hydrogelcontaining 1,3-propanediol obtained in Example 5 (Comparative), afterpreparation (A) and after freeze-drying and reconstitution (B), as afunction of time when the temperature increases from 4 to 37° C.

FIG. 7 shows the evolution of the elastic modulus G′ (storage modulus)and of the viscous modulus G″ (loss modulus) of the transparent hydrogelcontaining mannitol obtained in Example 6, after freeze-drying andreconstitution, as a function of time when the temperature increasesfrom 4 to 37° C.

FIG. 8 shows the evolution of the elastic modulus G′ (storage modulus)and of the viscous modulus G″ (loss modulus) of the transparent hydrogelcontaining glycerol obtained in Example 7, as a function of time whenthe temperature increases from 4 to 37° C.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

It is to be noted that in the present description and claims, theexpression “thermosetting” in connection with the composition of thepresent invention means that temperature does not induce the gelation ofthe composition but acts rather as a catalyst which dramaticallyshortens the gelation time when risen.

It is also to be noted that in the present description, the expression“hydrogel” or “hydrogel of the present invention” is used instead of“composition of the present invention” when appropriate.

It is still to be noted that in the present description and claims, theterm “neutralized” means a pH of 6.7-7.1.

According to the present invention, the aqueous thermosettingneutralized chitosan composition forming a phosphate-free transparenthydrogel at a temperature higher than 5° C. comprises a reacetylatedchitosan neutralized with an hydroxylated base and a complexing agentselected from polyoses and polyols derived from polyoses.

The average molecular weight (Mw) of the reacetylated chitosan comprisedin the composition of the present invention is typically not lower than100 kDa.

Molecular weight of chitosan may be determined by asymmetrical flowfield-flow fractionation (AFFF) coupled to multiangle light scattering(MALS) as reported for example by B. Wittgren and K.-G. Wahlund inJournal of Chromatography A 760:205-215 (1997).

Reacetylated chitosan having a Mw typically not lower than 100 KDa isparticularly appropriate for use in the present invention because itallows the formation of a thermosetting composition forming a firmhydrogel.

Preferably, the reacetylated chitosan used in the present invention hasa Mw not lower than 200 kDa.

The upper limit of the Mw of the reacetylated chitosan used in thepresent invention depends on the amount of the reacetylated chitosancomprised in the composition of the present invention and is determinedby the ease of administration, which depends on the chosen application.

Reacetylated chitosan used in the present invention must have adeacetylation degree of 40-70% which means that the chitosan comprises40 to 70% of D-glucosamine units and 60 to 30% of neutralN-acetyl-D-glycosamine units, respectively.

The deacetylation degree of chitosan may be determined by NuclearMagnetic Resonance such as described in the literature by Lavertu etal., Journal of Pharmaceutical and Biomedical Analysis 32: 1149-1158(2003).

If deacetylation degree of the reacetylated chitosan is lower than 40%,the reacetylated chitosan becomes a polymer close to chitin that isinsoluble in acidic conditions and consequently not usable in thepresent invention.

If deacetylation degree of the reacetylated chitosan is higher than 70%,the reacetylated chitosan does not allow the preparation of acomposition forming a phosphate-free transparent hydrogel.

Preferably, the deacetylation degree of the reacetylated chitosancomprised in the composition of the present invention is from 45 to 65%.

A reacetylated chitosan having a molecular weight typically not smallerthan 100 kDa and a deacetylation degree of 40-70% for use in the presentinvention may be prepared for example according to the process disclosedin WO-A-2005/097871 or may be obtained from Novamatrix (Oslo, Norway).

The amount of the reacetylated chitosan comprised in the composition ofthe present invention must be from 0.1 to 5.0 w/w %, based on the totalcomposition.

An amount of reacetylated chitosan lower 0.1 w/w % does not allow theformation of a hydrogel and an amount of reacetylated chitosan higherthan 5.0 w/w % induces the formation of a composition too difficult toinject.

The amount of reacetylated chitosan comprised in the composition of thepresent invention will be chosen depending on the Mw of the chitosan andon the aimed application.

Preferably, the amount of the reacetylated chitosan comprised in thecomposition of the present invention is from 0.5 to 3.0 w/w %, based onthe total composition.

The amount of the complexing agent comprised in the composition of thepresent invention must be from 1 to 30 w/w %, based on the totalcomposition, and will depend on the concentration and molecular weightof the reacetylated chitosan as well as on the required gelation timeand degree of consistency of the hydrogel.

The amount of the complexing agent comprised in the composition of thepresent invention is preferably from 5 to 15 w/w %, based on the totalcomposition.

Said complexing agent selected from polyoses and polyols derived frompolyoses comprised in the composition of the present invention allows tomodulate the properties of the hydrogel, such as gelation time andviscosity of the hydrogel.

In one preferred embodiment of the present invention, the complexingagent which may be used in the present invention is a polyose, morepreferably a polyose selected from monosaccharides and disaccharides.

As preferred examples of monosaccharides which may be used in thepresent invention, there may be cited D-glucose (also called dextrose),fructose and tagatose, which are known as excipients for pharmaceuticalcompositions according to European, US or Japanese pharmacopoeias.

As preferred examples of disaccharides which may be used in the presentinvention, there may be cited trehalose, sucrose, maltose and lactosewhich are known as excipients for pharmaceutical compositions accordingto European, US or Japanese pharmacopoeias, with trehalose beingparticularly preferred.

As other examples of polyoses which may be used in the presentinvention, there may be cited polysaccharides selected from polydextoseand amylose which are known as excipients for pharmaceuticalcompositions.

In another preferred embodiment of the present invention, the complexingagent which may be used in the present invention is a polyol derivedfrom polyose (also called sugar alcohol) selected from glycerol,mannitol, sorbitol, xylitol, erythritol, lactitol and maltitol, whichare known as excipients for pharmaceutical compositions according toEuropean, US or Japanese pharmacopoeias, with glycerol beingparticularly preferred.

The compositions of the present invention may be freezed for storagewhile preserving their thermogelling properties and have to be thawed at4° C. before their use.

Further, the compositions of the present invention, except thosecomprising glycerol as the complexing agent, may be advantageouslyfreeze-dried to obtain a lyophilizate for facilitated storage anddistribution, and reconstituted by the addition of cooled water to thelyophilizate under stirring at 4° C. before their use, while preservingtheir thermogelling properties.

The composition of the present invention may be prepared according to aprocess forming part of the present invention.

In step a) of said process, the reacetylated chitosan having a molecularweight typically not smaller than 100 kDa, preferably not smaller than200 kDa, a deacetylation degree of 40-70%, preferably 45-65%, issolubilized in an aqueous HCl medium and after complete dissolution ofchitosan, the temperature of the chitosan solution is cooled down to atemperature lower than 5° C., for example in an ice bath.

Then, in step b) of said process, the pH of the cooled chitosan solutionis neutralized until the pH 6.7-7.1, preferably pH 6.8, by addingdropwise, under stirring at a temperature lower than 5° C., the requiredamount of an aqueous solution containing a hydroxylated base previouslycooled to a temperature lower than 5° C.

A higher pH is not appropriate since it would induce the precipitationof chitosan.

According to said process, the hydroxylated base used for neutralizationis preferably NaOH.

Inadequate stirring or too fast addition of aqueous hydroxylated baseinduces the precipitation of the chitosan.

In step c) of said process, the complexing agent selected from polyosesand polyols derived from polyoses is added during or after thesolubilization step a), or before, during or after the neutralizationstep b).

It is pointed out that the description referring to chitosan, complexingagent and amounts thereof in connection with the composition of thepresent invention also applies in connection with the process of thepresent invention.

The process for preparing the composition of the present invention mayfurther comprise, if required, a step of sterilizing the reacetylatedchitosan before the step a) of solubilization. To obtain a sterilehydrogel, the preparation is performed under aseptic conditions (e.g.under a laminar flow) and every added solution is previously filteredthrough a 0.22 μm filter or steam-sterilized.

For example, sterilization may be performed by radiation or ideally bysteam sterilization of reacetylated chitosan suspended in water, asdescribed by Yen (Yen S.F. et al., 1998, U.S. Pat. No. 5,773,608).

The process for preparing the composition of the present invention mayfurther comprise, if required a step of freezing said composition forfacilitated storage.

In this case, the freezed composition has to be thawed at 4° C. beforeits use.

In a particularly preferred embodiment, the process according to thepresent invention may be completed by further freeze-drying thecomposition of the present invention, except the composition of thepresent invention containing glycerol as the complexing agent, to obtaina lyophilizate forming part of the present invention.

Said lyophilizate may be conveniently stored and distributed for medicaluse, and may be reconstituted by the addition of cold water understirring at 4° C.

When the temperature of the thermosetting neutralized chitosancomposition of the present invention is increased, for example afteradministration, thermogelation occurs leading to the formation of aphosphate-free, transparent firm hydrogel. The higher is thetemperature, the shorter is the gelation time.

According to the present invention, the composition of the presentinvention may be advantageously used as a drug delivery system and inview of its specific properties, may be advantageously used for thepreparation of an injectable formulation.

Further, since the lyophilizate of the present invention preserves itsthermogelling properties after reconstitution, it may be advantageouslyused for the preparation of a drug delivery system and for thepreparation of an injectable formulation.

In order to demonstrate the improved elastic properties of thephosphate-free transparent chitosan hydrogels of the present invention,rheological measurements of various hydrogels according to the presentinvention and comparative hydrogels were performed according to thefollowing method, unless otherwise indicated.

Viscoelastic properties of hydrogels were determined immediately afterpreparation of the hydrogels using a Rheostress 1 (Haake, Karlsruhe,Germany) using a cone/plate device (diameter 60 mm, angle 4°).Temperature was controlled with a thermostatic bath Haake DC 30 and acooling device Haake K10 (Haake, Karlsruhe, Germany) coupled with therheometer. Hydrogels were placed between the cone and plate (cooled downat 4° C.) and measured after 10 minutes. All measurements were performedin the linear viscoelastic range and G′ (storage modulus) and G″ (lossmodulus) were determined under a constant deformation (γ=0.05) at 1.00Hz for 180 minutes. The temperature was increased from 4 to 37° C. at6.6° C./min over the first 5 minutes, and maintained at 37° C. over thefollowing 175 minutes. Evaporation of water leading to drying ofhydrogels was minimized by use of a cover surrounding the cone/platedevice.

The following hydrogels have been tested

(1) the hydrogel of the present invention obtained in Example 1,containing 2 w/w % of chitosan (DD=47%) obtained from Novamatrix and 8w/w % trehalose, and the comparative hydrogel without trehalose (seeFIG. 2);

(2) the hydrogel of the present invention obtained in Example 1,containing 2 w/w % of chitosan (DD=47%) obtained from Novamatrix and 8w/w % trehalose, after preparation and after freezing-thawing accordingto Example 2 (see FIGS. 3A and 3B);

(3) the hydrogel of the present invention obtained in Example 1,containing 2 w/w % of chitosan (DD=47%) obtained from Novamatrix and 8w/w % trehalose, after freeze-drying and reconstitution according toExample 3, and the comparative hydrogel without trehalose (see FIG. 4);

(4) the hydrogel of the present invention obtained in Example 4,containing 0.9% w/w of chitosan (DD=61%) obtained according toPreparation Example 1, and 5 w/w % trehalose, and the comparativehydrogel without trehalose (see FIG. 5);

(5) a comparative hydrogel obtained in Example 5 (Comparative),containing 1 w/w % chitosan (DD 47%) obtained according to PreparationExample 2 and 10 w/w % 1,3-propanediol, after preparation and afterfreeze-drying and reconstitution (see FIGS. 6A and 6B);

(6) the hydrogel of the present invention obtained in Example 6,containing 0.9 w/w % chitosan (DD=61%) obtained according to PreparationExample 1, and 5 w/w % mannitol, after freeze-drying and reconstitution(see FIG. 7);

(7) the hydrogel of the present invention obtained in Example 7,containing 2 w/w % chitosan (DD 47%) obtained from Novamatrix and 10%glycerol, and the comparative hydrogel without glycerol (see FIG. 8).

FIGS. 2-8 show the evolution of the elastic modulus G′ (storage modulus)and of the viscous modulus G″ (loss modulus) of the tested hydrogels asa function of time when temperature increases from 4 to 37° C. The onsetof incipient formation of the gel network, which defines the gelationtime, is given by the time of crossover of G′ and G″.

As shown in FIG. 2, addition of a polyose such as trehalose according tothe present invention increases the G′ and G″ values of the hydrogel ascompared with the same hydrogel without trehalose.

FIG. 3A shows the viscoelastic properties of the hydrogel just after itspreparation (as reported in FIG. 2) while FIG. 3B shows the viscoelasticproperties of the same hydrogel after freezing and thawing.

As shown in FIG. 3B, thermogelling properties of the composition of thepresent invention are maintained after freezing and thawing.

FIG. 4 shows the viscoelastic properties of the hydrogel reported inFIG. 2 after freeze-drying and reconstitution.

As shown in FIG. 4, thermogelling properties of the composition of thepresent invention are maintained after freeze-drying and reconstitution.

As shown in FIG. 5, the composition of the present invention containingtrehalose shows a gelification point of 30 minutes, whereas the sameformulation containing no trehalose forms a gel after 150 minutes.

As shown in FIGS. 6A and 6B, the comparative composition containing1,3-propanediol forms an hydrogel when the viscoelastic properties aremeasured after its preparation (FIG. 6A) but does not preserve itsthermogelling properties after lyophilization and reconstitution asindicated by the absence of a gelification point in FIG. 6B.

As shown in FIG. 7, the composition of the present invention containingmannitol preserves its thermogelling properties after lyophilization andreconstitution, as indicated by the presence of a gelification point.

As shown in FIG. 8, the composition of the present invention containingglycerol forms an hydrogel, as indicated by the presence of agelification point whereas the same formulation containing no glycerolshows no gelification point after 180 minutes.

The following examples are intended to illustrate the present invention.However, they cannot be considered in any case as limiting the scope ofthe present invention.

EXAMPLES

In the following examples, the deacetylation degree of chitosans wasdetermined by Nuclear Magnetic Resonance (NMR) such as described in theliterature by Lavertu et al., Journal of Pharmaceutical and BiomedicalAnalysis 32: 1149-1158 (2003).

The molecular weight of chitosans was determined by asymmetrical flowfield-flow fractionation (AFFF) coupled to multiangle light scattering(MALS), as follows:

-   -   Fractionation of the chitosan solution (2 mg/ml in acetate        buffer pH 4.5) was performed in a trapezoidal channel, 26.5 cm        in length and 350 μm in height, connected to an Eclipse F system        (Wyatt Technology Europe, Dembach, Germany). The bottom of the        channel was lined with a regenerated cellulose membrane with a        10 kDa cut-off (Microdyn-Nadir GmbH, Wiesbaden, Germany). The        elution medium consisted of acetate buffer pH 4.5. The channel        flow was set to 1 ml/min and the injection flow to 0.2 ml/min.        The separation started with a focus flow of 1 ml/min for 3        minutes and was followed by a cross flow of 0.2 ml/min for 15        minutes. A Dawn EOS multi-angle light scattering detector (Wyatt        Technology, Santa Barbara, USA) and a refractive index (RI)        detector (Waters differential refractometer, Milford, Mass.,        USA) were coupled online with the field-flow fractionation        channel. The light scattering detector was equipped with a GaAs        laser (wavelength: 690 nm) and eighteen detectors. Scattered        light was collected at angles comprised between 14 and 163        degrees. The RI detector was calibrated with sodium chloride.        Data were collected and analysed with the Astra version 4.90.08        software, using a refractive index increment (dn/dc) of 0.153        ml/g.

In the following examples, injectability of some compositions wasdetermined with a device composed of a vertical support for a 1.0 mlluer lock syringe filled with the hydrogel at 3° C. and a pan resting onthe piston of the syringe as shown in FIG. 1. A 27 G^(1/2)×0.5 inchesneedles was fixed on the syringe which was positioned in the support. Amass (500 gr or 1 kg) was placed on this pan and the time necessary forthe composition to be expelled from the syringe was measured.

In the following Examples 1-3 and 7, the reacetylated chitosan used forthe preparation of the hydrogel is a reacetylated chitosan obtained fromNovamatrix (batch FU-507-03), with a DD of 47% (measured by NMR) and anaverage molecular weight (Mw) of 3600 kDa (measured by AFFF-MALS).

In the following Examples 4 and 6, the reacetylated chitosans used forthe preparation of the hydrogels are reacetylated chitosans preparedaccording to Preparation Example 1.

In the following Example 5, the reacetylated chitosan used for thepreparation of the hydrogel is reactetylated chitosan prepared accordingto Preparation Example 2.

PREPARATION EXAMPLE 1 Preparation of a Reacetylated Chitosan “Fagal Lot21” Having a DD of 61% According to the Method Disclosed inWO-A-2005/097871

25.5 gr of chitosan flakes (Sigma-aldrich, Saint Louis, Mo., USA,product number 41,941-9, batch 14418LB) were dissolved in 1 liter of asolution of acetic acid 10% and methanol (50/50) for one hour understirring. 550 ml of methanol were added. After 2 hours stirring, themixture was filtered through a 100 μm filter to eliminate insolubleparticles. The viscous solution was then dialyzed (Spectra/Por® 1Dialysis Membranes 6,000-8,000 MWCO, n^(o)132665, Spectrum Laboratories,Rancho Dominguez, USA) against deionized water for 72 hours, with dailychange of the water. The solution was then filtered through a 5 μmfilter.

Under stirring, 400 ml of NH₄OH 0.2M/methanol (50/50) were added toinduce precipitation. After 1 hour stirring, the suspension was filteredthrough a 100 μm filter. The precipitate was washed with methanol untilneutral. The purified chitosan obtained was dried in the presence ofSilicagel, under vacuum, at room temperature and protected from light.

10 g of this purified chitosan were dissolved in 500 ml acetic acid10%/methanol (50/50). The mixture was stirred for 1 hour and allowed tostand overnight. 400 ml of methanol were added. The solution was stirredfor several hours and allowed to stand overnight. 150 ml of methanolwere added and the chitosan solution was cooled down to a temperaturelower than 5° C. using an ice bath. A solution made of 2.4 ml aceticanhydride and 200 ml methanol was cooled down to a temperature lowerthan 5° C. and added dropwise to the chitosan solution, under vigorousmechanical stirring. This solution containing homogeneously reacetylatedchitosan was kept under stirring at a temperature lower than 5° C. forone hour to ensure complete reaction, and allowed to stand overnight atroom temperature. To eliminate salts produced during reacetylation andto further eliminate insoluble particles, this viscous solution wasdialyzed against deionized water for 12 days (same dialysis membranes asabove) with daily change of the water. The chitosan viscous solution wasthen filtered through a 5 μm filter. 200 ml of NH₄OH 0.2M/methanol(50/50) were added under stirring to induce chitosan precipitation.After 4 hours stirring, the chitosan was passed through a 100 μm filterand washed with methanol. Finally, the homogeneously reacetylatedchitosan was dried in the presence of Silicagel, under vacuum, at roomtemperature and protected from light.

The obtained reacetylated chitosan had a DD of 61% (measured by NMR) andan average molecular weight (Mw) of 7900 kDa (measured by AFFF-MALS).

PREPARATION EXAMPLE 2 Preparation of a Reacetylated Chitosan “Fagal Lot25” Having a DD of 47% According to the Method Disclosed inWO-A-2005/097871

Chitosan flakes (Sigma-aldrich, Saint Louis, Mo., USA, product number41,941-9, batch 14418LB) were purified as in Preparation Example 1.Chitosan was then reacetylated with a solution made of 2.0 ml aceticanhydride and 200 ml methanol according to the same procedure as inExample Preparation 1.

The obtained reacetylated chitosan had a DD of 47% (measured by NMR).

Example 1 Preparation of a Composition According to the PresentInvention, Containing 2% w/w of Reacetylated Chitosan (DD=47%_(RMN))Provided by Novamatrix and 8 w/w % Trehalose

700 mg of reacetylated chitosan provided by Novamatrix (batchFU-507-03), with a DD of 47% (measured by RMN) were autoclaved insuspension in water at a concentration of 4% (w/w). 145 μl HCl wereadded and the suspension was kept under stirring for 18 h at roomtemperature to allow complete chitosan solubilization. 3.09 g oftrehalose were solubilized in 6.5 ml NaOH 0.15M. This solution wascooled down in an ice bath and added dropwise under stirring to thecooled down chitosan solution. The pH of the gel was then adjusted to6.8 by dropwise addition of cooled down diluted NaOH. Finally, coldwater was added to obtain a total mass of 35 g. The transparent hydrogelobtained showed an increase of its viscoelastic behaviour following timeat 37° C., as illustrated in FIG. 2 and FIG. 3A and had a gelation oftime of 111 min. The injectability measurement gave 30 seconds with akilogram mass.

Example 2 Freezing-Thawing the Hydrogel Obtained in Example 1

10 gr of the preparation of Example 1 was frozen in liquid nitrogen andkept at −20° C. It was then thawed at 4° C. before its rheologicalproperties were determined. As shown in FIG. 3B, the thermogellingproperties were maintained.

Example 3 Freezing-Drying the Hydrogel Obtained in Example 1

10 gr of the preparation of Example 1 was frozen in liquid nitrogen andkept at −20° C. before being lyophilized for 24 hours with an EdwardsModulyo Freeze dryer (plate at −50° C., vacuum of 10⁻¹ mbar). Thelyophilizate obtained was reconstituted by the addition of cold waterunder stirring, at 4° C. As shown in FIG. 4, the thermogellingproperties were maintained.

Example 4 Preparation of a Composition According to the PresentInvention, Containing 0.9% w/w of Reacetylated Chitosan “Fagal Lot 21”(DD=61%_(RMN)) Obtained According to the Preparation Example 1 and 5 w/w% Trehalose

270 mg of reacetylated chitosan prepared according to PreparationExample 1 and having a DD of 61% (measured by RMN) were solubilized in15 ml HCl 0.1N under stirring for 18 hours at room temperature. 1.66 gof trehalose dihydrate were solubilized in 8 ml NaOH 0.15M. Thissolution was cooled down in an ice bath and added dropwise understirring, to the cooled down chitosan solution. The pH of the gel wasthen adjusted to 6.8 by dropwise addition of cooled down diluted NaOH.Finally, cold water was added to obtain a total mass of 30 g. Thetransparent hydrogel obtained showed an increase of its viscoelasticbehaviour following time at 37° C., as illustrated in FIG. 5. Theformulation containing trehalose showed a gelification point after 30minutes, whereas the formulation containing no trehalose formed a gelafter 150 minutes. The injectability measurement gave 30 seconds with a500 gr mass.

Example 5 (Comparative) Preparation of a Comparative CompositionContaining 1% w/w of Reacetylated Chitosan “Fagal Lot 25” (DD=47%_(RMN))Obtained According to the Preparation Example 2 and 10 w/w %1,3-propanediol

200 mg of reacetylated chitosan prepared according to PreparationExample 2 and having a DD of 47% (measured by RMN) were solubilized in10 ml HCl 0.1N under stirring for 18 hours at room temperature. 2 g ofcooled down 1,3-propanediol were added to the solubilized chitosan. ThepH of the gel was then adjusted to 6.8 by dropwise addition of cooleddown diluted NaOH. Finally, cold water was added to obtain a total massof 20 g. The transparent hydrogel obtained showed an increase of itsviscoelastic behaviour following time at 37° C., as illustrated in FIG.6A.

When frozen (at −80° C.), lyophilized (for 24 hours) and reconstitutedwith cold water under stirring at 4° C., the preparation obtained wasnot any longer injectable (27G^(1/2), a kilogram mass) in theinjectability measurement test. Moreover, it did not present anythermogelling properties, as shown in FIG. 6B.

Example 6 Preparation of a Composition According to the PresentInvention, Containing 0.9% w/w of Reacetylated Chitosan “Fagal Lot 21”(DD=61%_(RMN)) obtained according to the Preparation Example 1 and 5 w/w% mannitol

630 mg of the reacetylated chitosan obtained according to PreparationExample 1 were solubilized in 35 ml HCl 0.1N under stirring for 18 hoursat room temperature. This chitosan solution was cooled down to around 5°C. before adding 3.5 g of mannitol. The pH of the gel was then adjustedto 6.8 by dropwise addition of cooled down diluted NaOH. Finally, coldwater was added to obtain a total mass of 70 g. 10 g of this preparationwere frozen in liquid nitrogen and kept at −20° C. before beinglyophilized for 24 hours with an Edwards Modulyo Freeze dryer (plate at−50° C., vacuum of 10⁻¹ mbar). The lyophilizate obtained wasreconstituted by the addition of cold water under stirring, at 4° C. Thetransparent hydrogel obtained showed an increase of its viscoelasticbehaviour following time at 37° C., with a gelation time of 1 hour, asillustrated in FIG. 7.

Example 7 Preparation of a Composition According to the PresentInvention, Containing 2% w/w of Reacetylated Chitosan (DD=47%_(RMN))Provided by Novamatrix and 10 w/w % of Glycerol

700 mg of reacetylated chitosan obtained from Novamatrix were autoclavedin suspension in water at a concentration of 4% (w/w). After coolingdown the suspension at room temperature, 145 μl HCl were added and themixture was kept under stirring for 18 h to allow complete chitosansolubilization. The chitosan solution was cooled down to around 5° C.using an ice bath and 3.5 g of cooled down glycerol were added. The pHof the gel was then adjusted to 6.8 by dropwise addition of cooled downdiluted NaOH. Finally, cold water was added to obtain a total mass of 35g. The transparent hydrogel containing glycerol showed an increase ofits viscoelastic behaviour following time at 37° C. with a gelation timeof 135 minutes, as illustrated in FIG. 8, whereas the formulationcontaining no glycerol showed no gelification point after 180 minutes.

1. An aqueous thermosetting neutralized chitosan composition forming a phosphate-free transparent hydrogel at a temperature higher than 5° C., said composition comprising 0.1 to 5.0 w/w %, based on the composition, of a reacetylated chitosan having a molecular weight greater than or equal to 100 kDa and a deacetylation degree of 40 to 70%, the chitosan neutralized with a hydroxylated base, and 1 to 30 w/w %, based on the composition, of a complexing agent selected from the group consisting of polyoses and polyols derived from polyoses.
 2. The aqueous thermosetting neutralized chitosan composition according to claim 1, wherein the reacetylated chitosan is comprised in an amount of 0.5 to 3.0 w/w %, based on the composition.
 3. The aqueous thermosetting neutralized chitosan composition according to claim 1, wherein the deacetylation degree of the reacetylated chitosan is 45 to 65%.
 4. The aqueous thermosetting neutralized chitosan composition according to claim 1, wherein the molecular weight of the reacetylated chitosan is greater than or equal to 200 kDa.
 5. The aqueous thermosetting neutralized chitosan composition according to claim 1, wherein the complexing agent is comprised in an amount of 5 to 15 w/w %, based on the composition.
 6. The aqueous thermosetting neutralized chitosan composition according to claim 1, wherein the complexing agent is a polyose.
 7. The aqueous thermosetting neutralized chitosan composition according to claim 6, wherein the polyose is selected from the group consisting of monosaccharides and disaccharides.
 8. The aqueous thermosetting neutralized chitosan composition according to claim 7, wherein the polyose is a monosaccharide selected from the group consisting of D-glucose, fructose and tagatose.
 9. The aqueous thermosetting neutralized chitosan composition according to claim 7, wherein the polyose is a disaccharide selected from the group consisting of trehalose, sucrose, maltose and lactose.
 10. The aqueous thermosetting neutralized chitosan composition according to claim 9, wherein the disaccharide is trehalose.
 11. The aqueous thermosetting neutralized chitosan composition according to claim 1, wherein the complexing agent is a polyol derived from a polyose selected from the group consisting of glycerol, mannitol, sorbitol, xylitol, erythritol, lactitol and maltitol.
 12. The aqueous thermosetting neutralized chitosan composition according to claim 1 wherein the composition is a lyophilizate obtained by freeze-drying the aqueous thermosetting neutralized chitosan composition, the composition containing glycerol as the complexing agent.
 13. A process for producing the aqueous thermosetting neutralized chitosan composition according to claim 1, which comprises the steps of: a) solubilizing an appropriate amount of a reacetylated chitosan having a molecular weight greater than or equal to 100 kDa and a deacetylation degree of 40-70%, in an aqueous HCl medium and cooling a chitosan solution to a temperature lower than 5° C.; b) neutralizing the chitosan solution obtained in step a) by adding an aqueous hydroxylated base previously cooled to a temperature lower than 5° C. to the chitosan solution until the solution of chitosan exhibits a 6.7-7.1; c) adding a complexing agent selected from the group consisting of polyoses and polyols derived from polyoses during or after the solubilization step a), or before during or after the neutralization step b), and d) optionally, freezing the aqueous thermosetting neutralized chitosan composition.
 14. The process according to claim 13, further comprising a step of sterilizing the reacetylated chitosan before the step a) of solubilization.
 15. The process according to claim 13, wherein in step b), the hydroxylated base is NaOH.
 16. A process according to claim 13, which comprises freeze-drying the aqueous thermosetting neutralized chitosan composition, wherein the composition excludes glycerol as the complexing agent.
 17. A method of using the aqueous thermosetting neutralized chitosan composition of claim 1, as a drug delivery system.
 18. The method of using the aqueous thermosetting neutralized chitosan composition according to claim 17, for the preparation of an injectable formulation.
 19. A method of using the lyophilizate according to claim 12, for the preparation of a drug delivery system.
 20. A method of using the lyophilizate according to claim 12, for the preparation of an injectable formulation. 